Vitamin c and doxycycline: a synthetic lethal combination therapy for eradicating cancer stem cells (cscs)

ABSTRACT

The present disclosure relates to compounds and methods of eradicating cancer stem cells by combining inhibitors of oxidative metabolism and glycolytic metabolism. Also described are compounds and methods of identifying a combination of inhibitors of oxidative metabolism and glycolytic metabolism to treat cancer stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/488,489, filed Apr. 21, 2017, the entirety of whichis incorporated herein by reference.

FIELD

The present disclosure relates to methods of eradicating cancer stemcells by combining inhibitors of oxidative metabolism and glycolyticmetabolism.

BACKGROUND

Researchers have struggled to develop new anti-cancer treatments.Conventional cancer therapies (e.g. irradiation, alkylating agents suchas cyclophosphamide, and anti-metabolites such as 5-Fluorouracil) haveattempted to selectively detect and eradicate fast-growing cancer cellsby interfering with cellular mechanisms involved in cell growth and DNAreplication. Other cancer therapies have used immunotherapies thatselectively bind mutant tumor antigens on fast-growing cancer cells(e.g., monoclonal antibodies). Unfortunately, tumors often recurfollowing these therapies at the same or different site(s), indicatingthat not all cancer cells have been eradicated. Relapse may be due toinsufficient chemotherapeutic dosage and/or emergence of cancer clonesresistant to therapy. Hence, novel cancer treatment strategies areneeded.

Advances in mutational analysis have allowed in-depth study of thegenetic mutations that occur during cancer development. Despite havingknowledge of the genomic landscape, modern oncology has had difficultywith identifying primary driver mutations across cancer subtypes. Theharsh reality appears to be that each patient's tumor is unique, and asingle tumor may contain multiple divergent clone cells. What is needed,then, is a new approach that emphasizes commonalities between differentcancer types. Targeting the metabolic differences between tumor andnormal cells holds promise as a novel cancer treatment strategy. Ananalysis of transcriptional profiling data from human breast cancersamples revealed more than 95 elevated mRNA transcripts associated withmitochondrial biogenesis and/or mitochondrial translation. Sotgia etal., Cell Cycle, 11(23):4390-4401 (2012). Additionally, more than 35 ofthe 95 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs).Proteomic analysis of human breast cancer stem cells likewise revealedthe significant overexpression of several mitoribosomal proteins as wellas other proteins associated with mitochondrial biogenesis. Lamb et al.,Oncotarget, 5(22):11029-11037 (2014). Functional inhibition ofmitochondrial biogenesis using the off-target effects of certainbacteriostatic antibiotics or OXPHOS inhibitors provides additionalevidence that functional mitochondria are required for the propagationof cancer stem cells.

There exists a need in the art for novel anticancer strategies, newcompounds with broad-spectrum antibiotic activity, and compounds toreduce the effects of aging. The “endosymbiotic theory of mitochondrialevolution” can be used as the basis for the development of therapies totreat drug-resistance that is characteristic of both tumor recurrenceand infectious disease, and such therapies may have the additionalbenefit of slowing the aging process.

In view of the foregoing, it is therefore an objective of thisdisclosure to demonstrate that mitochondrial biogenesis plays a criticalrole in the propagation and maintenance of many cancers. It is also anobjective of this disclosure to demonstrate that the combination ofmitochondrial-targeting compounds and glycolysis-targeting compounds maybe used to eradicate cancer stem cells (CSCs) by metabolically“starving” the CSCs. It is also an objective of this disclosure topresent methods for identifying and using the combination ofmitochondrial-targeting compounds and glycolysis-targeting compounds fortherapeutic purposes.

The inventors analyzed phenotypic properties of CSCs that could betargeted across a wide range of cancer types and identified a strictdependence of CSCs on mitochondrial biogenesis for the clonal expansionand survival of CSCs. Previous work by the inventors demonstrated thatdifferent classes of FDA-approved antibiotics, and in particulartetracyclines, such as doxycycline, and erythromycin have an off-targeteffect of inhibiting mitochondrial biogenesis. Such compounds could haveefficacy for eradicating CSCs. Unfortunately, when used alone theseantibiotics do not eradicate all CSCs; rather, these antibioticsmetabolically synchronize a surviving CSC sub-population from oxidativemetabolism to glycolytic metabolism, resulting in metabolicinflexibility. The present disclosure demonstrates that the use ofcompounds that metabolically target the antibiotic-resistant CSCsub-population in combination with the mitochondrial-targeting compoundsmay be used to eradicate CSCs.

SUMMARY

The present disclosure relates to methods of treating cancer byadministering to a patient in need thereof of a pharmaceuticallyeffective amount of an inhibitor of oxidative metabolism and aninhibitor of glycolytic metabolism. Inhibitors of oxidative metabolismmay include members of tetracycline family and the erythromycin family.Members of the tetracycline family include tetracycline, doxycycline,tigecycline, minocycline, chlortetracycline, oxytetracycline,demeclocycline, lymecycline, meclocycline, methacycline,rolitetracycline, chlortetracycline, omadacycline, and sarecycline.Members of the erythromycin family include erythromycin, azithromycin,and clarithromycin. Inhibitors of glycolytic may be selected frominhibitors of glycolysis, inhibitors of OXPHOS, and inhibitors ofautophagy. Inhibitors of glycolysis include 2-deoxy-glucose, ascorbicacid, and stiripentol. Inhibitors of OXPHOS include atoravaquone,irinotecan, sorafenib, niclosamide, and berberine chloride. Inhibitorsof autophagy include chloroquine.

The present disclosure also relates to methods of identifying acombination of inhibitors of oxidative metabolism and glycolyticmetabolism to treat cancer stem cells, the method comprising:chronically treating cancer stem cells with at least one inhibitor ofoxidative metabolism; confirming the chronically treated cancer stemcells manifest a glycolytic phenotype; further treating the chronicallytreated cancer stem cells with at least one inhibitor of glycolyticmetabolism; and confirming inhibition of glycolytic metabolism. Thesemethods may include treating MCF7 cells. The at least one inhibitor ofoxidative metabolism may be selected from the tetracycline family and/orat least one member of the erythromycin family. The member of thetetracycline family may be selected from the group comprising at leastone of tetracycline, doxycycline, tigecycline, minocycline,chlortetracycline, oxytetracycline, demeclocycline, lymecycline,meclocycline, methacycline, rolitetracycline, chlortetracycline,omadacycline, and sarecycline. The member of the erythromycin family maybe selected from the group comprising at least one of erythromycin,azithromycin, and clarithromycin. Confirming that the chronicallytreated cancer stem cells manifest a glycolytic phenotype may includeperforming metabolic flux analysis and/or performing label-free unbiasedproteomics. Metabolic flux analysis may include measuring oxygenconsumption rates, measuring extracellular acidification rates, andmeasuring mammosphere formation. Performing label-free unbiasedproteomics may include measuring relative changes to mitochondrialprotein levels and/or measuring relative changes to glycolytic enzymelevels. The at least one inhibitor of glycolytic metabolism may beselected from the group comprising an inhibitor of glycolysis, aninhibitor of OXPHOS, and an inhibitor of autophagy. Inhibitors ofglycolysis include 2-deoxy-glucose, ascorbic acid, and stiripentol.Inhibitors of OXPHOS include atoravaquone, irinotecan, sorafenib,niclosamide, and berberine chloride. Inhibitors of autophagy includechloroquine. Confirming inhibition of glycolytic metabolism may includemeasuring mammosphere formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show the effects of doxycycline treatment on mitochondrialmass. FIGS. 1A-C show the effects of increasing concentrations ofdoxycycline over time on mitochondrial mass (1A shows effects of 12.5 μMdoxycycline treatment, 1B shows effects of 25 μM doxycycline treatment,1C shows effects of 50 μM doxycycline treatment). FIG. 1D showsrepresentative plots illustrating increased mitochondrial mass ofdoxycycline-resistant MCF7 cells as compared to untreated MCF7 cells.FIG. 1E shows confirmation of mitochondrial mass by immuno-blot analysiswith specific antibodies directed against TOMM20, an established markerof mitochondrial mass.

FIG. 2A illustrates the effects of doxycycline treatment on oxygenconsumption rate (OCR) over time in MCF7 cells. FIG. 2B shows theeffects of doxycycline treatment on OCR for basal respiration, protonleak, ATP-linked respiration, maximal respiration, and spare respiratorycapacity.

FIG. 3 shows the effects of doxycycline treatment on extracellularacidification rate (ECAR) over time in MCF7 cells. FIG. 4 shows theeffects of doxycycline treatment on ECAR for glycolysis, glycolyticreserve, and glycolytic reserve capacity.

FIGS. 5A and 5C show the effects of doxycycline treatment on CSC markersALDH and CD44⁺/CD24^(low) activity, respectively. FIGS. 5B and 5D-Eshows the effects of doxycycline treatment on CD24 and CD44 usingfluorescence activated cell sorting (FACS).

FIGS. 6A-B show the effects of Atovaquone and Cloroquine on thepropagation of doxycycline-treated and untreated MCF7 cells.

FIG. 7A shows the effects of doxycycline treatment on cell proliferationusing an EdU incorporation assay. FIG. 7B shows the reduction of the EdUpositive population in doxycycline-treated MCF7 cells as compared toMCF7 cells using FACS. FIG. 7C shows the effects of doxycyclinetreatment on cell migration using a wound healing assay. FIG. 7Dprovides images of cells tested using the scratch assay. FIG. 7E-F showthe effects of doxycycline on ERK1/2 and AKT Ser 473 phosphorylation,respectively.

FIG. 8 outlines a method for targeting mitochondrial activity andglycolysis to target and eradicate CSCs.

FIGS. 9 and 10 show the effects of glycolysis inhibitors 2-deoxy-glucose(2 DG) (FIG. 9) and ascorbic acid (FIG. 10) on mammosphere formation indoxycycline-treated MCF7 cells.

FIGS. 11A-F show the effects of Stiripentol (FIG. 11A), Irinotecan (FIG.11B), Sorafenib (FIG. 11C), Berberine Chloride (FIG. 11D), andNiclosamide (FIG. 11E-F) on mammosphere formation in doxycycline-treatedMCF7 cells.

FIG. 12 outlines the method by which mitochondrial function andglycolysis are blocked to inhibit CSC propagation.

DESCRIPTION

The following description illustrates embodiments of the presentapproach in sufficient detail to enable practice of the presentapproach. Although the present approach is described with reference tothese specific embodiments, it should be appreciated that the presentapproach can be embodied in different forms, and this description shouldnot be construed as limiting any appended claims to the specificembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the present approach to those skilled in the art.

The mitochondrial ribosome is an untapped gateway for treating a numberof afflictions, ranging from cancer to bacterial and fungal infectionsto aging. Functional mitochondria are required for the propagation ofCSCs. Inhibiting mitochondrial biogenesis in cancer cells impedes thepropagation of those cells. Mitochondrial inhibitors therefore representa new class of anti-cancer therapeutics. In some cases, however, asurviving cancer cell sub-population may metabolically synchronizetoward a glycolytic phenotype. The inventors hypothesized that combiningan inhibitor of glycolysis with a mitochondrial inhibitor may present amethod for eradicating CSCs.

To test this hypothesis, the inventors generated a cancer cellsub-population metabolically synchronized toward a glycolytic phenotypeby chronically treating MCF7 cells with the FDA-approved antibioticdoxycycline, a mitochondrial inhibitor. Briefly, MCF7 cells wereobtained from ATCC and cultured in DMEM (Sigma Aldrich). MCF7 cellsresistant to doxycycline (MCF7-DoxyR cells) were selected by a stepwiseexposure to increasing concentrations of doxycycline. Specifically, MCF7cells were initially exposed to 12.5 μM doxycycline, followed by 3 weeksof treatment with 25 μM doxycycline, followed by 3 weeks of treatmentwith 50 μM doxycycline. The doxycycline-resistant MCF7 cells were thenroutinely maintained in regular medium supplemented with 25 μMDoxycycline. It should be appreciated that other cancer cell lines andother mitochondrial (oxidation) inhibitors may be used.

The present approach further involves methods of analyzing the effectsof chronic treatment on cells by considering changes to mitochondrialmass. The inventors measured mitochondrial mass by FACS analysis, but itshould be appreciated that other methods known in the art to measuremitochondrial mass may be used. Briefly, cells were stained withMitoTracker Deep Red (Life Technologies), which localizes tomitochondria regardless of mitochondrial membrane potential. Cells wereincubated with pre-warmed MitoTracker staining solution (diluted inPBS/CM to a final concentration of 10 nM) for 30-60 min at 37° C. Allsubsequent steps were performed in the dark. Cells were washed in PBS,harvested, re-suspended in 300 μL of PBS and then analyzed by flowcytometry (Fortessa, BD Bioscience, Calif., USA). Data analysis wasperformed using FlowJo software. Extracellular acidification rates(ECAR) and real-time oxygen consumption rates (OCR) for MCF7 cells weredetermined using the Seahorse Extracellular Flux (XFe-96) analyzer(Seahorse Bioscience, Mass., USA). 15,000 MCF7 and MCF7 DoxyR cells perwell were seeded into XFe-96 well cell culture plates for 24 h. Then,cells were washed in pre-warmed XF assay media (or for OCR measurement,XF assay media supplemented with 10 mM glucose, 1 mM Pyruvate, 2 mML-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175μL/well of XF assay media at 37 C, in a non-CO₂ incubator for 1 hour.During the incubation time, 5 μL of 80 mM glucose, 9 μM oligomycin, and1 M 2-deoxyglucose (for ECAR measurement) or 10 μM oligomycin, 9 μMFCCP, 10 μM Rotenone, 10 μM antimycin A (for OCR measurement), wereloaded in XF assay media into the injection ports in the XFe-96 sensorcartridge. Data set was analyzed by XFe-96 software after themeasurements were normalized by protein content (SRB). All experimentswere performed three times independently. FIGS. 2A-D show thatMCF7-DoxyR cells exhibit a significant increase in mitochondrial mass(by ˜1.3- to 1.7-fold), as compared to acute treatment with doxycyclineat the same drug concentration. The overall increase in mitochondrialmass was confirmed by immuno-blot analysis with specific antibodiesdirected against TOMM20, a well-established marker of mitochondrial mass(FIG. 1E).

The present approach also includes methods of analyzing the effects ofchronic treatment on cells by considering changes to oxygen consumptionrates. FIGS. 3A-B show that MCF7-DoxyR cells also exhibited asignificant reduction in oxygen consumption rates (OCR), as compared tocontrol MCF7 cells. Reduced OCR suggests that ATP levels are severelydepleted in the MCF7-DoxyR cells. Conversely, glycolysis wassubstantially increased in the MCF7-DoxyR cells as measured by the ECAR(extracellular acidification rate) (FIGS. 3 and 4). Therefore, thegenerated DoxyR cells were mainly glycolytic, thus validating thehypothesis that a sub-population of CSCs may survive and developdoxycycline-resistance by adopting a purely glycolytic phenotype.

The present approach further involves methods of analyzing the effectsof chronic treatment on cells by examining relative changes of CSCmarkers and functional CSC activity using, for example, mammosphere,proliferation, and cell migration assays. For example, aldehydedehydrogenase (ALDH) activity and CD44/CD24 levels are routinely used asmarkers to identify breast CSCs. ALDH activity may be assessed by FACSanalysis. The ALDEFLUOR kit (StemCell Technologies, Mass., USA) may beused to isolate the population with high ALDH enzymatic activity.Briefly, 1×105 MCF7 and MCF7 DoxyR cells may be incubated in 1 mlALDEFLUOR assay buffer containing ALDH substrate (5 μl/ml) for 40minutes at 37° C. In each experiment, a sample of cells may be stainedunder identical conditions with 30 μM of diethylaminobenzaldehyde(DEAB), a specific ALDH inhibitor, as a negative control. TheALDEFLUOR-positive population may be established according to themanufacturer's instructions and evaluated in 3×10⁴ cells. Data analysismay be performed using FlowJo software. An Anoikis assay may be used todetermine CD24/CD44 expression. Briefly, MCF7 and MCF7 DoxyR cells maybe seeded on low-attachment plates to enrich for the CSC population.Under these conditions, the non-CSC population undergoes anoikis (a formof apoptosis induced by a lack of cell-substrate attachment) and CSCsare believed to survive. The surviving CSC fraction may be analyzed byFACS analysis. Briefly, 1×10⁵ MCF7 and MCF7 DoxyR monolayer cells may beseeded for 48 h in 6-well plates. Then, cells may be trypsinized andseeded in low-attachment plates in mammosphere media. After 10 h, cellsmay be spun down and incubated with CD24 (IOTest CD24-PE, BeckmanCoulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodiesfor 15 minutes on ice. Cells may be rinsed twice and incubated withLIVE/DEAD dye (Fixable Dead Violet reactive dye; Life Technologies) for10 minutes. Samples may then be analyzed by FACS. Only the livepopulation, as identified by the LIVE/DEAD dye staining, may be analyzedfor CD24/CD44 expression using FlowJo software. FIGS. 5A-D showMCF7-DoxyR cells have a substantial increase in ALDH activity andCD44/CD24 levels. Notably, ALDH activity and CD44/CD24 levels do notreflect active CSC activity.

To more directly assess functional CSC activity, mammosphere formationassays may be used. Briefly, a single cell suspension of MCF7 or MCF7DoxyR cells may be prepared using enzymatic (1× Trypsin-EDTA, SigmaAldrich), and manual disaggregation (25-gauge needle). Cells may beplated at a density of 500 cells/cm2 in mammosphere medium(DMEM-F12/B27/20-ng/ml EGF/PenStrep) in nonadherent conditions, inculture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA,Sigma), in the presence of Atovaquone (FIG. 6A) or Chloroquine (FIG.6B). Cells may be grown for 5 days and maintained in a humidifiedincubator at 37° C. at an atmospheric pressure in 5% (v/v) carbondioxide/air. After 5 days for culture, spheres larger than 50 μm may becounted using an eye piece graticule, and the percentage of cells platedwhich formed spheres may be calculated. Mammosphere assays may beperformed in triplicate and repeated three times independently. FIG. 6shows that MCF7-DoxyR cell propagation is significantly more sensitiveto Atovaquone, as compared with control MCF7 CSCs. Specifically,treatment with 1 μM Atovaquone inhibited the CSC propagation ofMCF7-DoxyR cells by more than 85%. Similarly, Chloroquine inhibitedpropagation by more than 75% at 25 μM and by more than 90% at 50 μM.These results suggest it is possible to target the propagation of DoxyRCSCs using existing FDA-approved OXPHOS and autophagy inhibitors.Therefore, increases in CSC marker levels do not necessarily reflectfunctional increases in CSC propagation.

To determine functional effects on doxycycline on CSC propagation,proliferation may be measured by determining relative levels of EdUincorporation (EdU refers to the alkyne-containing thymidine analog(5-ethynyl-2′-deoxyuridine) which is incorporated into DNA during activeDNA synthesis). For example, 48 h after seeding MCF7 and MCF7-DoxyRcells were subjected to a proliferation assay using Click-iT Plus EdUPacific Blue Flow Cytometry Assay Kit (Life Technologies) customized forflow cytometry. Briefly, cells were treated with 10 μM EdU for 2 hoursand then fixed and permeabilized. EdU was detected afterpermeabilization by staining cells with Click-iT Plus reaction cocktailcontaining the Fluorescent dye picolylazide for 30 min at roomtemperature. Samples were then washed and analyzed using flow cytometer.Background values were estimated by measuring non-EdU labeled, butClick-iT stained cells. Data were analyzed using FlowJo software. FIGS.7A-B show MCF7-DoxyR cells have a reduced ability to proliferate by morethan 60%, as measured using EdU-incorporation.

The present approach further involves methods of analyzing the effectsof chronic treatment on functionality by considering cell migration.MCF7-DoxyR cells also show a clear defect in cell migration, with agreater than 50% reduction, as observed using the standard “scratchassay” (FIG. 7C-D). To determine cell migration, MCF7 and MCF7 DoxyRcells were allowed to grow in regular growth medium until they were70-80% confluent. Next, to create a scratch of the cell monolayer, ap200 pipette tip was used. Cells were washed twice with PBS and thenincubated at 37° C. in regular medium for 24 h. The migration assay wasevaluated using Incucyte Zoom (Essen Bioscience). The rate of migrationwas measured by quantifying the % of wound closure area, determinedusing the software ImageJ, according to the formula: % of woundclosure=[(At=0 h−At=Δh)/At=0 h]×100%. Data was represented as themean±standard error of the mean (SEM), taken over ≥3 independentexperiments, with ≥3 technical replicates per experiment. Statisticalsignificance was measured using the t-test. P≤0.05 was consideredsignificant.

Phosphorylation levels of proteins involved in cell signaling, such asERK and AKT, may be investigated to further determine cell phenotype. Todetermine ERK and AKT phosphorylation, MCF7 and MCF7 DoxyR cells proteinlysates were electrophoresed through a reducing SDS/10% (w/v)polyacrylamide gel, electroblotted onto a nitrocellulose membrane andprobed with primary antibodies against phosphorylated AKT (Ser 473) andATK (Cell Signaling), phopshorylated ERK 1/2 (E-4), ERK2 (C-14), TOMM20(F-10) and β-actin (C2) (all purchased from Santa Cruz Biotechnology).Proteins were detected by horseradish peroxidase-linked secondaryantibodies and revealed using the SuperSignal west pico chemiluminescentsubstrate (Fisher Scientific). FIGS. 7E-F show MCF7-DoxyR cells havesignificant reductions in ERK-activation and AKT-activation. Thesefindings demonstrate that MCF7-DoxyR cells have a quiescent glycolyticcell phenotype.

To further validate the functional observations from metabolic fluxanalysis, unbiased label-free proteomics analysis may be conducted.Briefly, cell lysates may be prepared for trypsin digestion bysequential reduction of disulphide bonds with TCEP and alkylation withMMTS. Peptides may be extracted and prepared for LC-MS/MS. All LC-MS/MSanalyses may be performed on an LTQ Orbitrap XL mass spectrometer(Thermo Scientific, San Jose, Calif.) coupled to an Ultimate 3000 RSLCnano system (Thermo Scientific, formerly Dionex, NL). Xcalibur raw datafiles acquired on the LTQ-Orbitrap XL may be directly imported intoProgenesis LCMS software (Waters Corp., Milford, Mass., USA, formerlyNon-Linear Dynamics, Newcastle upon Tyne, UK) for peak detection andalignment. Data may be analyzed using the Mascot search engine. Fivetechnical replicates may be analyzed for each sample type.

TABLE 1 Key Mitochondrial-Related Proteins are Down-Regulated inDoxy-Resistant MCF7 Cells. Fold-reduction Symbol Description(Down-regulation) Mitochondrial proteins encoded by mitochondrial DNAMT-ND3 NADH-ubiquinone oxidoreductase chain 3 (Complex I) 35.07 MT-CO2Cytochrome c oxidase subunit 2 (Complex IV) 19.26 MT-ATP8 ATP synthaseprotein 8 (Complex V) 6.42 MT-ATP6 ATP synthase subunit 6 (Complex V)5.08 Mitochondrial proteins encoded by nuclear DNA NDUFS1NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial 12.53 NNTNAD(P) transhydrogenase, mitochondrial 10.49 SSBP1 Single-strandedDNA-binding protein, mitochondrial 9.27 NDUFB8 NADH dehydrogenase 1 betasubcomplex subunit 8, mitochondrial 8.5 CKMT1A Creatine kinase U-type,mitochondrial 7.49 TFAM Transcription factor A, mitochondrial 6.89 COX7CCytochrome c oxidase subunit 7C, mitochondrial 5.4 COX7A2 Cytochrome coxidase subunit 7A2, mitochondrial 5.34 SDHB Succinate dehydrogenaseiron-sulfur subunit, mitochondrial 4.86 COX5B Cytochrome c oxidasesubunit 5B, mitochondrial 4.83 CKMT2 Creatine kinase S-type,mitochondrial 4.78 COQ6 Ubiquinone biosynthesis monooxygenase COQ6,mitochondrial 4.71 HYOU1 Hypoxia up-regulated protein 1 4.55 CHDHCholine dehydrogenase, mitochondrial 4.42 NDUFV1 NADH dehydrogenase[ubiquinone] flavoprotein 1, mitochondrial 4.31 PUS1 tRNA pseudouridinesynthase A, mitochondrial 4.28 OXCT1 Succinyl-CoA:3-ketoacid coenzyme Atransferase 1, mitochondrial 4.17 TOMM6 Mitochondrial import receptorsubunit TOM6 4.15 ACAA2 3-ketoacyl-CoA thiolase, mitochondrial 4.04 NFU1NFU1 iron-sulfur cluster scaffold homolog, mitochondrial 3.96 CPT1ACarnitine O-palmitoyltransferase 1, liver isoform 3.52 UQCRC1 Cytochromeb-c1 complex subunit 1, mitochondrial 3.51 PRKDC DNA-dependent proteinkinase catalytic subunit 3.43 MDH2 Malate dehydrogenase, mitochondrial3.3 ACSF3 Acyl-CoA synthetase family member 3, mitochondrial 3.29 FHFumarate hydratase, mitochondrial 3.27 PDHX Pyruvate dehydrogenaseprotein X component, mitochondrial 3.23 BDH1 D-beta-hydroxybutyratedehydrogenase, mitochondrial 3.16 NDUFS3 NADH dehydrogenase iron-sulfurprotein 3, mitochondrial 3.16 MMAB Cob(I)yrinic acid a,c-diamideadenosyltransferase, mitochondrial 3.12 DARS2 Aspartate--tRNA ligase,mitochondrial 3 SUCLA2 Succinyl-CoA ligase [ADP-forming] subunit beta,mitochondrial 2.91 ABAT 4-aminobutyrate aminotransferase, mitochondrial2.83 LACTB Serine beta-lactamase-like protein LACTB, mitochondrial 2.81CHDH Choline dehydrogenase, mitochondrial 2.78 GLS Glutaminase kidneyisoform, mitochondrial 2.77 TOMM34 Mitochondrial import receptor subunitTOM34 2.76 NDUFA10 NADH dehydrogenase 1 alpha subcomplex subunit 10,mitochondrial 2.7 MUL1 Mitochondrial ubiquitin ligase activator of NFKB1 2.6 UQCRC2 Cytochrome b-c1 complex subunit 2, mitochondrial 2.54COX7A2L Cytochrome c oxidase subunit 7A-related protein, mitochondrial2.54 SLC25A24 Calcium-binding mitochondrial carrier protein SCaMC-1 2.51NDUFA9 NADH dehydrogenase 1 alpha subcomplex subunit 9, mitochondrial2.5 GLUL Glutamine synthetase 2.5 PDHA1 Pyruvate dehydrogenase E1subunit alpha, somatic, mitochondrial 2.5 SDHA Succinate dehydrogenaseflavoprotein subunit, mitochondrial 2.48 NDUFS8 NADH dehydrogenaseiron-sulfur protein 8, mitochondrial 2.42

Table 1 summarizes mitochondrial-related proteins that aredown-regulated in MCF7-DoxyR cells. Down-regulated proteins includethose encoded by mitochondrial DNA (mt-DNA) and nuclear DNA (nuc-DNA).For example, the cellular levels of MT-ND3, MT-CO2, MT-ATP6, and MT-ATP8are reduced 5-35 fold. Such reductions may inactivate or impair thefunctioning of Complex I, IV, and V. Similarly, more than 45nuclear-encoded mitochondrial proteins, such as NDUFS1, NDUFB8, andCOX7C, are reduced between 2-12 fold. Loss of mt-DNA-encoded proteins ischaracteristic of the inhibition of mitochondrial protein translation.

In contrast, the levels of glycolytic enzymes, such as PGM1, LDHA,ALDOC, and GAPDH, increased 2-7 fold, as is shown in Table 2. Similarly,enzymes associated with glycogen metabolism increased 3-4 fold (Table2).

TABLE 2 Enzymes Related to Glycolysis and Glycogen Metabolism areUp-Regulated in Doxy-Resistant MCF7 Cells. Fold-Increase SymbolDescription (Up-regulation) Glycolytic enzymes PGM1 Phosphoglucomutase-17.16 LDHA L-lactate dehydrogenase A 7.09 ALDOC Fructose-bisphosphatealdolase C 3.44 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 3.06GPD1L Glycerol-3-phosphate dehydrogenase 1-like 2.72 protein ALDOAFructose-bisphosphate aldolase A 2.71 PFKP ATP-dependent6-phosphofructokinase, 2.69 platelet type PGK1 Phosphoglycerate kinase 12.64 GPI Glucose-6-phosphate isomerase 2.46 PKM Pyruvate kinase 2.1Glycogen metabolism GYS1 Glycogen [starch] synthase, muscle 4.11 PYGMGlycogen phosphorylase, muscle form 3.45 PYGL Glycogen phosphorylase,liver form 3.39

Table 3 shows that markers of hypoxia, including myoglobin andhemoglobin (alpha/delta), were elevated, thus further suggesting apredominantly glycolytic phenotype. Consistent with an increase inAldefluor activity, several ALDH gene products were increased, such asALDH1A3. Increased ALDH activity may reflect the cells' preferencetowards glycolysis, as ALDH isoforms contribute significantly to theglycolytic pathway.

TABLE 3 Markers of Hypoxia and Cancer Stem Cells are Up-regulated inDoxy-Resistant MCF7 Cells. Fold-Increase Symbol Description(up-regulation) Hypoxia markers MB Myoglobin 5.86 HBA1 Hemoglobinsubunit alpha 3.46 HBD Hemoglobin subunit delta 1.81 ALDH gene isoformsALDH1A3 Aldehyde dehydrogenase family 1 1,681.32 member A3 ALDH1A2Retinal dehydrogenase 2 5.22 ALDH5A1 Succinate-semialdehyde 3.87dehydrogenase, mitochondrial ALDH18A1 Delta-1-pyrroline-5-carboxylatesynthase 2.75 ALDH16A1 Aldehyde dehydrogenase family 16 2.04 member A1Other cancer stem cell (CSC) markers RGAP2 SLIT-ROBO RhoGTPase-activating 2.8 protein 2 CD44 CD44 antigen 2.09

Table 4 shows that ten mitochondrial ribosomal proteins (MRPs) increasedbetween 1.5-3 fold. Increases in MRPs may explain the increase in themitochondrial mass discussed above and in FIG. 2.

TABLE 4 A Subset of Mitochondrial Ribosomal Proteins (MRPs) areIncreased in Doxy-Resistant MCF7 Cells. Fold-Increase Symbol Description(up-regulation) Small subunit MRPS25 28S ribosomal protein S25,mitochondrial 3.02 MRPS9 28S ribosomal protein S9, mitochondrial 1.69MRPS18C 28S ribosomal protein S18c, mitochondrial 1.58 Large subunitMRPL10 39S ribosomal protein L10, mitochondrial 2.9 MRPL12 39S ribosomalprotein L12, mitochondrial 2.21 MRPL46 39S ribosomal protein L46,mitochondrial 2.13 MRPL53 39S ribosomal protein L53, mitochondrial 2.13MRPL37 39S ribosomal protein L37, mitochondrial 2.05 MRPL19 39Sribosomal protein L19, mitochondrial 1.95 MRPL15 39S ribosomal proteinL15, mitochondrial 1.94

Table 5 illustrates that cellular ribosomal proteins may bedown-regulated, between 1.8-9 fold. Such downregulation may drivedecreases in cellular protein synthesis due to mitochondrial energydeficits, resulting in a quiescent metabolic phenotype.

TABLE 5 A Subset of Cellular Ribosomal Proteins are Decreased inDoxy-Resistant MCF7 Cells. Fold-reduction Symbol Description(Down-regulation) Small subunit RPS15 40S ribosomal protein S15 2.12RPS21 40S ribosomal protein S21 2.08 RPS4X 40S ribosomal protein S4, Xisoform 2.06 RPS23 40S ribosomal protein S23 1.82 Large subunit RPL3460S ribosomal protein L34 9.85 RPL3 60S ribosomal protein L3 6.39 RPLP260S acidic ribosomal protein P2 3.68 RPL10A 60S ribosomal protein L10a2.28 RPL27A 60S ribosomal protein L27a 2.06 RPL8 60S ribosomal proteinL8 1.93 RPL22L1 60S ribosomal protein L22-like 1 1.82 Other RSL1D1Ribosomal L1 domain-containing 3.08 protein 1

The DoxyR cells acquire a predominantly glycolytic phenotype to escapethe anti-mitochondrial effects of doxycycline. The inventorshypothesized that the DoxyR cells are metabolically synchronized, aremetabolically inflexible, and therefore should be sensitive toadditional metabolic stressors or perturbations, allowing them to beeliminated completely. The inventors hypothesized that additionalmetabolic stressors could be added using metabolic inhibitors targetingglycolysis, OXPHOS, and/or autophagy. This “two-hit” metabolic scheme isillustrated in FIG. 8. DoxyR cells are identified S801, and arecharacterized as metabolically synchronized and metabolically inflexibleS802. DoxyR cells may be exposed to one or more metabolic stressor, suchas metabolic inhibitors targeting glycolysis, OXPHOS, and/or autophagy,thereby reducing doxycycline resistance S803. Then doxycycline (oranother antimitochondrial compound) may be administered to eradicate thecells during their reduced resistance S804.

To test the “two-hit” metabolic hypothesis, the inventors tested theeffects of Atovaquone, an FDA-approved OXPHOS inhibitor which targetsmitochondrial Complex III, and Chloroquine, an autophagy inhibitor.Atovaquone and Chloroquine are normally used clinically for thetreatment and prevention of malaria, a parasitic infection. It should beappreciated by one of skill in the art that other OXPHOS and autophagyinhibitors may be selected. A list of exemplary metabolic inhibitors ispresented below.

TABLE 6 Combination Therapies Doxycycline Plus Doxycycline PlusDoxycycline Plus OXPHOS Inhibitor Glycolysis Inhibitor AutophagyInhibitor Atovaquone 2-Deoxy-glucose (2-DG) Chloroquine IrinotecanAscorbic acid Sorafenib Stiripentol Niclosamide Berberine Chloride

The present approach further involves methods of testing the efficacy ofglycolysis inhibitors on CSC propagation using 2-deoxy-glucose (2-DG)and Vitamin C (ascorbic acid). It should be appreciated that otherglycolysis inhibitors may be used. Treatment with 2-DG inhibited thepropagation of DoxyR CSCs by more than 90% at 10 mM and 100% at 20 mM(FIGS. 9 and 10). Vitamin C may be more potent than 2-DG, as itinhibited DoxyR CSC propagation by more than 90% at 250 μM and 100% at500 μM (FIGS. 9 and 10). The IC-50 for Vitamin C in this experiment wasbetween 100 to 250 μM, which is within the known achievable blood levelswhen Vitamin C is taken orally. The inventors previously showed that theIC-50 for Vitamin C was 1 mM for MCF7 CSC propagation. Bonuccelli et al,Oncotarget 8:20667-20678 (2017). Therefore, DoxyR CSCs may beapproximately 4-10-fold more sensitive to Vitamin C than control MCF7CSCs under identical assay conditions.

The present approach further involves methods of testing the efficacy ofglycolysis inhibitors on CSC propagation using other clinically-approveddrugs that functionally behave as OXPHOS inhibitors (Irinotecan,Sorafenib, Niclosamide) or glycolysis inhibitors (Stiripentol) onmammosphere formation. Briefly, MCF7 DoxyR cells were cultured in lowattachment plates and treated with Vehicle or increasing concentrationsof the lactate dehydrogenase (LDH) inhibitor Stiripentol (2 μM to 100μM) (FIG. 11A) or the OXPHOS inhibitors Irinotecan (500 nM to 80 μM)(FIG. 11B), Sorafenib (500 nM to 40 μM) (FIG. 11C), Berberine Chloride(500 nM to 10 μM) (FIG. 11D) and Niclosamide (FIG. 11E-F) for 5 daysbefore counting. Independent experiments were performed in triplicate.FIG. 11A-F shows that Niclosamide was most potent at inhibiting DoxyRCSC propagation (IC-50 ˜100 nM), followed by Irinotecan (IC-50 ˜500 nM),Sorafenib (IC-50 ˜0.5-1 μM), Berberine (IC-50 ˜1 μM) and Stiripentol(IC-50 ˜10-50 μM).

Mitochondrial biogenesis inhibitors include tetracyclines (e.g.,tetracycline, doxycycline, tigecycline, and minocycline); erythromycins(e.g., eyrthromycin, azithromycin, and clarithromycin); pyrviniumpamoate; atovaquone; bedaquiline; irinotecan; sorafenib; niclosamide;berberine; stiripentol; chloroquine; etomoxir; perhexiline;mitoriboscins, such as those disclosed in U.S. Provisional PatentApplication No. 62/471,688, filed Mar. 15, 2017, and Patent CooperationTreaty (PCT) Patent Application PCT/US2018/022403, filed Mar. 14, 2018,the entireties of which are incorporated herein by reference;mitoketoscins, such as those disclosed in U.S. Provisional PatentApplication No. 62/524,829, filed Jun. 26, 2017, the entirety of whichis incorporated herein by reference; mitoflavoscins, such as thosedisclosed in U.S. Provisional Patent Application No. 62/576,287, filedOct. 24, 2017, the entirety of which is incorporated herein byreference; TPP-compounds (e.g., 2-butene-1,4-bis-TPP), such as thosedisclosed in U.S. Provisional Patent Application No. 62/590,432, filedNov. 24, 2017, the entirety of which is incorporated herein byreference; mDIVI1, such as those disclosed in U.S. Provisional PatentApplication No. 62/608,065, filed Dec. 20, 2017, the entirety of whichis incorporated herein by reference; CAPE (caffeic acid phenyl ester);antimitoscins, such as those disclosed in 62/508,702, filed May 19,2017, the entirety of which is incorporated herein by reference;repurposcins such as those disclosed in U.S. Provisional PatentApplication No. 62/593,372, filed Dec. 1, 2017, the entirety of which isincorporated herein by reference; other known mitochondrial inhibitors.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. The invention includes numerousalternatives, modifications, and equivalents as will become apparentfrom consideration of the following detailed description.

It will be understood that although the terms “first,” “second,”“third,” “a),” “b),” and “c),” etc. may be used herein to describevarious elements of the invention should not be limited by these terms.These terms are only used to distinguish one element of the inventionfrom another. Thus, a first element discussed below could be termed aelement aspect, and similarly, a third without departing from theteachings of the present invention. Thus, the terms “first,” “second,”“third,” “a),” “b),” and “c),” etc. are not intended to necessarilyconvey a sequence or other hierarchy to the associated elements but areused for identification purposes only. The sequence of operations (orsteps) is not limited to the order presented in the claims.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification is controlling.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. Thus, the termconsisting essentially of” as used herein should not be interpreted asequivalent to “comprising.”

The term “about,” as used herein when referring to a measurable value,such as, for example, an amount or concentration and the like, is meantto encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount. A range provided herein for a measureable valuemay include any other range and/or individual value therein.

Having thus described certain embodiments of the present invention, itis to be understood that the invention defined by the appended claims isnot to be limited by particular details set forth in the abovedescription as many apparent variations thereof are possible withoutdeparting from the spirit or scope thereof as hereinafter claimed.

What is claimed is:
 1. A method of treating cancer comprisingadministering to a patient in need thereof a pharmaceutically effectiveamount of an inhibitor of oxidative metabolism and an inhibitor ofglycolytic metabolism.
 2. The method of claim 1, wherein the inhibitorof oxidative metabolism is selected from the group comprising at leastone member of tetracycline family and at least one member of theerythromycin family.
 3. The method of claim 2, wherein the member of thetetracycline family is selected from the group comprising at least oneof tetracycline, doxycycline, tigecycline, minocycline,chlortetracycline, oxytetracycline, demeclocycline, lymecycline,meclocycline, methacycline, rolitetracycline, chlortetracycline,omadacycline, and sarecycline.
 4. The method of claim 3, wherein themember of the tetracycline family is doxycycline.
 5. The method of claim2, wherein the member of the erythromycin family is selected from thegroup comprising at least one of erythromycin, azithromycin, andclarithromycin.
 6. The method of claim 1, wherein the inhibitor ofglycolytic metabolism is selected from the group comprising at least oneof an inhibitor of glycolysis, an inhibitor of OXPHOS, and an inhibitorof autophagy.
 7. The method of claim 6, wherein the inhibitor ofglycolysis is selected from the group comprising at least one of2-deoxy-glucose, ascorbic acid, and stiripentol.
 8. The method of claim6, wherein the inhibitor of OXPHOS is selected from the group comprisingat least one of atoravaquone, irinotecan, sorafenib, niclosamide, andberberine chloride.
 9. The method of claim 6, wherein the inhibitor ofautophagy is chloroquine.
 10. A method of identifying a combination ofinhibitors of oxidative metabolism and glycolytic metabolism to treatcancer stem cells, the method comprising: chronically treating cancerstem cells with at least one inhibitor of oxidative metabolism;confirming the chronically treated cancer stem cells manifest aglycolytic phenotype; further treating the chronically treated cancerstem cells with at least one inhibitor of glycolytic metabolism; andconfirming inhibition of glycolytic metabolism.
 11. The method of claim10, wherein the cancer stem cells are MCF7 cells.
 12. The method ofclaim 10, wherein the at least one inhibitor of oxidative metabolism isselected from the group comprising at least one member of tetracyclinefamily and at least one member of the erythromycin family.
 13. Themethod of claim 12, wherein the member of the tetracycline family isselected from the group comprising at least one of tetracycline,doxycycline, tigecycline, minocycline, chlortetracycline,oxytetracycline, demeclocycline, lymecycline, meclocycline,methacycline, rolitetracycline, chlortetracycline, omadacycline, andsarecycline.
 14. The method of claim 13, wherein the member of thetetracycline family is doxycycline.
 15. The method of claim 12, whereinthe member of the erythromycin family is selected from the groupcomprising at least one of erythromycin, azithromycin, andclarithromycin.
 16. The method of claim 10, wherein confirming thechronically treated cancer stem cells manifest a glycolytic phenotypecomprises at least one of performing metabolic flux analysis andperforming label-free unbiased proteomics.
 17. The method of claim 16,wherein performing metabolic flux analysis comprises at least one ofmeasuring oxygen consumption rates, measuring extracellularacidification rates, and measuring mammosphere formation.
 18. The methodof claim 16, wherein performing label-free unbiased proteomics comprisesat least one of measuring relative changes to mitochondrial proteinlevels and measuring relative changes to glycolytic enzyme levels. 19.The method of claim 10, wherein the at least one inhibitor of glycolyticmetabolism is selected from the group comprising an inhibitor ofglycolysis, an inhibitor of OXPHOS, and an inhibitor of autophagy. 20.The method of claim 19, wherein the inhibitor of glycolysis is selectedfrom the group comprising at least one of 2-deoxy-glucose, ascorbicacid, and stiripentol.
 21. The method of claim 19, wherein the inhibitorof OXPHOS is selected from the group comprising at least one ofatoravaquone, irinotecan, sorafenib, niclosamide, and berberinechloride.
 22. The method of claim 19, wherein the inhibitor of autophagyis chloroquine.
 23. The method of claim 10, wherein confirminginhibition of glycolytic metabolism comprises measuring mammosphereformation.
 24. A compound comprising a pharmaceutically effective amountof an oxidative metabolism inhibitor and a glycolytic metabolisminhibitor.
 25. The compound of claim 24, wherein the oxidativemetabolism inhibitor is selected from the group comprising at least onemember of tetracycline family and at least one member of theerythromycin family.
 26. The compound of claim 25, wherein the member ofthe tetracycline family is selected from the group comprising at leastone of tetracycline, doxycycline, tigecycline, minocycline,chlortetracycline, oxytetracycline, demeclocycline, lymecycline,meclocycline, methacycline, rolitetracycline, chlortetracycline,omadacycline, and sarecycline.
 27. The compound of claim 25, wherein themember of the tetracycline family is doxycycline.
 28. The compound ofclaim 25, wherein the member of the erythromycin family is selected fromthe group comprising at least one of erythromycin, azithromycin, andclarithromycin.
 29. The compound of claim 24, wherein the glycolyticmetabolism inhibitor is selected from the group comprising at least oneof an inhibitor of glycolysis, an inhibitor of OXPHOS, and an inhibitorof autophagy.
 30. The compound of claim 29, wherein the glycolysisinhibitor is selected from the group comprising at least one of2-deoxy-glucose, ascorbic acid, and stiripentol.
 31. The compound ofclaim 24, wherein the oxidative metabolism inhibitor is doxycycline, andthe glycolytic metabolism inhibitor is ascorbic acid.